Method for obtaining peptides

ABSTRACT

The present invention relates to a method of obtaining peptides from procaryotic and/or eucaryotic cells.

TECHNICAL FIELD

The present invention relates to a method of obtaining peptides fromprocaryotic and/or eucaryotic cells for their subsequent analysis.

The analysis of these peptides can indeed prove useful in a large numberof applications, particularly in biomarker research or bacterial peptideanalysis, for example within the framework of applications ofidentification, typing, detection of resistance and virulence markers,and this within the medical, pharmaceutical and agro-food fields.

STATE OF THE ART

The protocols of extraction and purification of peptides according tothe prior art are generally very lengthy. Moreover, these methods are,generally, manual and complex to implement.

International application WO 2006/031063 describes compositions intendedto hydrolyse polypeptides, this hydrolysis reaction being obtainedchemically by the use of a solution called “PCA”, comprising an acid(such as pure acetic acid), a water-miscible organic solvent (such asacetonitrile) and a reducing agent (such astris(2-carboxyethyl)phosphine). This chemical digestion is performed at99.9° C. for two hours. However WO 2006/031063 makes provision,optionally, for linking this step of chemical digestion to a prior stepof enzymatic digestion, possibly in order to increase the efficiency ofthe proteolysis. The enzymatic digestion step is performed at 37° C. for12 hours using a modified trypsin, subsequently to a thermaldenaturation step (90° C. for 20 minutes). The above-mentioned step ofchemical digestion is performed following this step of enzymaticdigestion (99.9° C. for 2 hours). The total treatment time of theprotein sample is therefore greater than 14 hours, which represents anindisputable major disadvantage. Moreover, WO 2006/031063 teaches theuse of a modified trypsin, more expensive than a conventional trypsin.

International application WO 2008/128029, for its part, discloses amethod of protein or peptide fragmentation in solution, said methodcomprising, subsequently to the initial step of enzymatic or chemicaldigestion, a series of additional steps of separation and fragmentation.The latter contribute on the one hand to increasing the time of thefractionation method overall and, on the other, require variousmanipulations, thus rendering the automation of this method extremelydifficult.

U.S. Pat. No. 7,622,273 describes a protocol intended to denature thepost-translationally modified polypeptides or proteins, these peptidesand proteins being directly bound to a protein microarray and saidprotocol comprising the steps of chemical treatment, enzymatic orchemical digestion and of subsequent identification of proteins on saidprotein microarrays. The step of chemical treatment comprises thedenaturation, the reduction and the alkylation of said proteins, whilethat of enzymatic digestion includes the deglycosylation and/ordephosphorylation of said proteins and the digestion of the latter bychemical or enzymatic proteolysis. All the reactions are performedsequentially on protein microarray. The step of chemical treatment lastsat least 3 hours and 15 minutes and that of enzymatic digestion twohours (cf. Example 1), i.e. a minimum duration of 5 hours and 15minutes. This, although less than the duration of the conventionalmethods—approximately 24 hours—nevertheless remains unsatisfactory,particularly having regard to the requirements inherent in the clinicalor pharmaceutical fields. What is more, the complex samples (plasma,urine, cerebrospinal fluid, etc.) can require fractionation or depletionstrategies to isolate the target proteins prior to the implementation ofthe protocol disclosed in this patent, which increases the totaltreatment duration of said samples accordingly.

International application WO 2011/130521 describes a method ofproteolytic digestion, which uses a range of pressure cycles (forexample from 5 to 35 kpsi, i.e. approximately from 344.74 bars to2413.16 bars) in order to reduce the duration of this method. Even ifthis duration seems shorter than the methods of the prior art, itnonetheless remains that the cumulated time corresponding to the stepsof reduction (10 mM of DTT at 37° C. for 1 hour) and of alkylation (50mM of iodoacetamide at ambient temperature, in darkness, for 45 minutes)cannot be less than 1 hour and 45 minutes. If to this is added the timeof lysis of the microorganisms (3 minutes at 4500 rpm), the time ofmanipulation of the lysate before the step of reduction (not specified)and that inherent in the dilutions of the solution before the step ofenzymatic digestion under pressure (also not specified), it is obviousthat the total duration of the protocol which is the object of WO2011/130521 cannot be less than 2 hours, which remains unsatisfactory.Due to the high pressures used, this method requires a complexapparatus, able to withstand these high pressures, difficult to operateand expensive. What is more, said method requires additional steps offiltration in order to reduce the very high concentrations of chaotropicagent (8M urea), prior to the step of enzymatic digestion in order notto risk inactivation of the enzyme used Indeed, when used at highconcentration (from 1M), the chaotropic agents used at the step ofdenaturation of the proteins—such as urea—will also denature the proteinstructure of the enzyme(s) used at the step of enzymatic proteolysis andwill have the consequence of totally or partially inactivating thisenzyme. In order to prevent this, it is necessary, before the step ofenzymatic proteolysis (enzymatic digestion), to perform additional stepsof dilution and/or of filtration, which requires an additional timelapse and makes the automation of the method as a whole more complex. Analternative consists in using genetically modified enzymes (for examplemodified trypsins) capable of performing protein digestion in thepresence of high concentrations of chaotropic agent(s). However, thesemodified enzymes have less rapid action kinetics than those of thenative enzymes and have a much higher purchase cost than these latter.

There therefore exists a need to develop a protocol permitting forobtaining of peptides from procaryotic and/or eucaryotic cells whichresolves all or part of the above-mentioned problems, i.e. an efficient,rapid, easily automatable protocol not requiring unnecessary steps ofdilution.

DISCLOSURE OF THE INVENTION

Thus an object of the present invention relates to a method of obtainingpeptides from procaryotic and/or eucaryotic cells, said methodcomprising the following steps:

-   -   a) lysis of the procaryotic and/or eucaryotic cells and recovery        of the proteins thus obtained,    -   b) denaturation of said proteins using at least one denaturing        agent,    -   c) alkylation of the denatured proteins using at least one        alkylating agent,    -   d) enzymatic proteolysis of the proteins obtained at the end of        step c) using at least one proteolytic enzyme,    -   e) recovery of the peptides obtained at the end of the step of        enzymatic proteolysis d),        in which the lysis of the procaryotic and/or eucaryotic cells in        step a) is lysis at a low concentration of chaotropic agent(s).

This method (protocol) of obtaining peptides is applicable toprocaryotic cells (for example to bacteria), to eucaryotic cells (human,animal, yeast, etc. cells) or to a mixture of procaryotic and eucaryoticcells.

According to a preferred embodiment, the method according to the presentinvention will be used in order to obtain peptides from procaryoticcells, preferably from bacteria (Gram+ or Gram−).

By “denaturing agent” is understood an agent—physical orchemical—capable of inducing a phenomenon of denaturation of proteinsand polypeptides; the latter leaving their native state and little bylittle losing their secondary, tertiary and quaternary structure. Thedenaturation can sometimes be reversible, the return to the native statethen being possible and the activity of the protein being restored.

The denaturation of the proteins is due to the sensitivity of the latterdepending on their physico-chemical environment. The proteins aredenatured when the interactions between the residues are disrupted by adenaturing agent. The covalent bonds between adjacent amino acids of thepolypeptide chain are not broken. Conversely, certain conditions ofdenaturation can cause the breaking of disulphide bonds betweennon-adjacent cysteine residues of the polypeptide chain which providethe overall stability of the quaternary structure of the protein.

As denaturing agent are principally distinguished:

a) physical agents, such as temperature; the increase of the temperatureindeed causes thermal agitation of the atoms of the molecule; thiscauses breakage of weak interactions like the hydrogen bonds, whichstabilise the spatial structure;

b) chemical agents, such as:

-   -   acids and bases; by modifying the surrounding pH they induce a        modification of the charges carried by the ionisable groups and        therefore damage the ionic and hydrogen bonds stabilising the        spatial structure of the protein;    -   chaotropic agents such as urea, guanidine salts (for example        guanidine hydrochloride or lithium perchlorate); used at high        concentrations, these compounds greatly weaken the hydrogen        bonds of the proteins (main low-energy bonds responsible for        maintaining the secondary, tertiary and quaternary structures of        the proteins);    -   thiol reducing agents such as 2-mercaptoethanol or        dithiothreitol (DTT); they permit cleaving of the disulphide        bridges and thus contribute to weakening the tertiary or        quaternary structure of the proteins; —detergents, which act by        modification of the interaction with the aqueous solvent (for        example sodium dodecyl sulphate—better known under the acronym        “SDS”).

Certain denaturing agents can prove non-reversible, such as the heavymetals (Pb, Hg, etc.) and certain acids (for example HNO₃,trichloroacetic acid, etc.

Of the chaotropic agents, a distinction is made, for the purposes of thepresent invention, between so-called “saline” chaotropic agents, such asthe salts of guanidine (or of guanidinium) and so-called “non-saline”chaotropic agents, such as urea.

By “lysis at low concentration of “chaotropic agent(s)”, is understoodlysis at a concentration of chaotropic agent(s) less than or equal to1M, preferably less than or equal to 100 mM.

Indeed, at such concentrations, so-called “chaotropic” agents no longerexercise denaturing activity on proteins and polypeptides, thus losingtheir chaotropic property.

This technical solution is in contrast to the conventional solutionspresented in the prior art, which generally employ chaotropic agents andmore particularly guanidine salts (such as guanidine hydrochloride,guanidine thiocyanate, etc.)—at generally very high concentrations, ofthe order of 6M. Such concentrations of chaotropic agents—andparticularly of salts (for example of guanidine salts)—greatly interferewith the enzymatic proteolysis, in particular when the latter isperformed using a serine protease such as trypsin. The fact, within theframework of the method of obtaining peptides according to the presentinvention, of performing lysis at a low concentration of chaotropicagent(s) not only permits improvement of the efficiency of theproteolysis step d) but also prevents having to have recourse toadditional filtration/dilution steps before said proteolysis step. Thetime saving achieved allows a significantly more rapid protocol to beobtained than those described in the state of the art, but which is alsomore easily automatable.

Moreover, this low concentration of chaotropic agent(s) allows the useof native proteolytic enzymes and does not require recourse togenetically modified proteolytic enzymes, which have less rapid actionkinetics than those of the native enzymes and have a much higherpurchase cost than the purchase cost of the latter.

According to a preferred embodiment, lysis is effected at a lowconcentration of salt(s), which means that the lysis is performed at asalt concentration less than or equal to 50 mM, preferably less than orequal to 30 mM.

Advantageously, the lysis at low concentration of chaotropic agent(s) isperformed in the absence of non-saline chaotropic agent(s) such as urea.Indeed, the method according to the present invention does not requirethe use of such non-saline chaotropic agents.

For lysis at a low concentration of chaotropic agent(s), it ispreferable to use the universal protocol of cellular lysis ofprocaryotes and/or eucaryotes described in international application WO02/10333 the whole of the content of which is incorporated by referencein the present patent application. This universal protocol consists in amethod of cellular lysis of procaryotes and/or of eucaryotes or ofsimultaneous cellular lysis of procaryotes and eucaryotes which consistsin adjusting at least three of the following parameters:

-   -   a percentage by mass of active beads (lysing beads) of small        diameter relative to active beads (lysing beads) of large        diameter less than or equal to 50%, and/or    -   a total mass of lysing (active) beads comprising a mixture or        otherwise of beads of small diameter and/or of beads of large        diameter, of between 50 and 100% relative to the total mass of        the biological sample treated, and/or    -   a duration of lysis of between 10 and 20 min, and/or    -   a number of non-lysing glass beads for driving lysing (active)        beads less than seven (7), and/or—a number of non-lysing iron        beads for driving lysing (active) beads of between five (5) and        fifteen (15), depending on the technique used:    -   sonication,    -   mechanical vortex, or    -   magnetic vortex.

Preferably, the lysing (active) beads of small diameter are of adiameter of between 90 and 150 μm and preferably approximately 100 μm,and the lysing (active) beads of large diameter are of a diameter ofbetween 400 and 600 μm and preferably approximately 500 μm.

Still preferably, the lysing (active) beads are made of glass.

According to a preferred embodiment, still such as indicated inapplication WO 02/10333, if the mechanical vortex technique is used, themethod consists in performing the lysis according to the followingparameters:

-   -   a duration of lysis of 11 to 20 min, preferably of 15 to 20 min        and still more preferably of 20 min,    -   a percentage of 100 μm diameter beads of less than 50%,        preferably less than 30%, and still more preferably of 20%,    -   a total mass of lysing (active) beads of greater than 60%,        preferably greater than 80%, and still more preferably of 100%        of the total mass of the biological sample treated, and    -   a number of glass beads less than seven (7) preferably equal to        one (1).

If the magnetic vortex technique is used, the method consists,preferably, in performing lysis according to the following parameters:

-   -   a duration of lysis of 12 to 20 min, preferably of 15 to 20 min        and still more preferably of 20 min,    -   a total mass of lysing (active) beads of 100 μm greater than 80%        and preferably of 100% of the total mass of the biological        sample treated, and    -   a number of iron beads of between five (5) to fifteen (15)        beads, preferably of ten (10) iron beads.

Generally, for the purposes of the present invention, any lysistechniques are used with a low concentration of chaotropic agent(s) notrequiring recourse to high pressures of the order of several hundreds oreven thousands of bars. Indeed, one of the objectives of the presentinvention is the elaboration of a protocol for obtaining peptides whichis efficient, rapid and can be implemented by means of a standardapparatus, without requiring the use of a specifically adapted apparatusto withstand high or even very high pressures, an apparatus which iscomplex to employ and extremely costly.

The method of obtaining peptides according to the present invention isimplemented at a pressure of from atmospheric pressure to a pressure ofapproximately one hundred bars. Preferably, said method is implementedunder a pressure of 100 bars, preferably less than 50 bars,advantageously less than 10 bars. According to a preferred embodiment,the method according to the present invention is performed atatmospheric pressure.

According to a preferred embodiment of the present invention, the lysisat a low concentration of chaotropic agent(s) is lysis by sonicationeffected by means of an ultrasound probe. Preferably this lysis bysonication is effected by means of said ultrasound probe in the presenceof a mixture of glass beads of 1000 μm and of zirconium beads of 100 μm.

Within the framework of lysis by sonication, preferably, the protocol isapplied for lysis by sonication such as described in internationalapplication WO 02/10333 (p. 3, 1. 18-26), i.e. using the followingparameters:

-   -   a duration of lysis of 9 to 20 min, preferably of 12 to 18 min        and still more preferably of 15 min,    -   a percentage of beads of 100 μm diameter of between 10 to 50%        preferably of between 20 and 30% and still more preferably of        20%, and    -   a total mass of lysing (active) beads of between 50 to 100% of        the total mass of the biological sample treated, preferably of        between 75 and 90% and preferably between 80 and 85%.

Concerning the apparatus and methodology of the protocol for lysis bysonication per se, in advantageous manner, an external sonotrode such asthe VialTweeter sonotrode marketed by the company Hielscher is used.Such a probe consists in a vibrating block drilled with holes in whichcan be inserted vials such as Eppendorf vials of 1.5 ml volume intowhich are introduced the cells to be lysed in suspension in a 3 mMBorate pH 8 buffer as well as the 1 mm glass and 100 μm zirconium beads(50 mg of each). The vial is closed and then the sonotrode activated for5 to 15 minutes, preferably 10 minutes, at an amplitude of between 50%and 100% of the nominal amplitude (between 5 and 10 watts for each vialdepending on its position in the block), preferably 100% and a cyclicratio of 40% to 60%, preferably of 50%.

The selection of a protocol for lysis by sonication is not in any caseand arbitrary choice. Much to the contrary, the applicant has, insurprising manner, discovered that the yield of the method of obtainingpeptides according to the present invention is better when the step oflysis a) was a step of lysis by sonication.

Moreover, the applicant has discovered that the omission of the step ofpreheating the ultrasound probe (activation of the probe lasting 1 h,which raises the temperature of the whole of the vibrating system to 95°C.) did not impair the efficiency of said method. This proves to be analtogether advantageous characteristic in that this step of preheatingof the ultrasound probe reduces its lifetime and requires an additionalcontribution of energy.

In consequence of which, in preferred manner, the ultrasound probe isnot subjected to a step of preheating prior to lysis by sonication.

Regarding the step of enzymatic proteolysis d), this latter is performedby action of a proteolytic enzyme, preferably a serine protease,advantageously selected from the group consisting in trypsin,chymotrypsin and elastase. Preferably, said proteolytic enzyme istrypsin.

This enzymatic proteolysis is particularly preferred relative to thephysico-chemical treatments (treatments with hydrogen peroxide, cyanogenbromide, trifluoroacetic acid, etc.) as it preserves the structure ofthe proteins more, proves easier to control and cleaves the peptidechains at specific sites (at the C-terminal of lysine and arginineresidues in the case of trypsin). By “enzymatic proteolysis” isunderstood the single or combined action of one or more enzymes underappropriate reaction conditions. The enzymes performing the proteolysis,called proteases, cleave the proteins in site-specific manner. Indeedeach protease generally recognises a specific sequence of amino acids inwhich this protease always performs the same cleavage. Certain proteasesrecognise a single amino acid or a sequence of two amino acids betweenwhich they perform a cleavage, other proteases recognise longersequences of amino acids. These proteases can be endoproteases orexoproteases. Among these known proteases can be cited, as described inthe document WO 2005/098071:

-   -   specific enzymes such as trypsin which splits the peptide bond        at the carboxylic group of the Arg and Lys residues, endolysin        which cleaves the peptide bond of the —CO group of the lysines,        chymotrypsin which hydrolyses the peptide bond at the carboxyl        group of the aromatic residues (Phe, Tyr and Trp), pepsin which        cuts at the NH2 group of the aromatic residues (Phe, Tyr and        Trp), V8 protease of the V8 strain of Staphylococcus aureus        which cleaves the peptide bond at the carboxyl group of the Glu        residue;    -   non-specific enzymes such as thermolysin derived from the        bacterium Bacillus thermoproteolyticus which hydrolyses the        peptide bond of the NH2 group of the hydrophobic amino acids        (Xaa-Leu, Xaa-Ile, Xaa-Phe), subtilisin and pronase which are        bacterial proteases which hydrolyse practically all the bonds        and can transform proteins into oligopeptides under controlled        reaction conditions (enzyme concentration and reaction        duration).

Several proteases can be used in a simultaneous manner, if their modesof action are compatible, or they can be used successively. Within theframework of the invention, the digestion of the sample is, preferably,performed by action of a protease enzyme having good cleavage siteselectivity, for example trypsin.

The obtaining of peptides by means of a chemical reagent or of aprotease can be obtained by simple reaction in solution. It can also beimplemented with a microwave oven or under pressure or with anultrasound device In these three last cases, the protocol can be muchmore rapid.

Of the peptides obtained by enzymatic digestion (enzymatic proteolysis),the specific peptides of the protein are called proteotypic peptides.The latter are easily detected by mass spectrometry or other appropriateanalytical techniques suited to the detection of such proteotypicpeptides. This represents an additional advantage relative to chemicalproteolysis, obtained by means of physico-chemical treatments.

Preferably, the denaturing agent is a thiol reducing agent, preferablyselected from 2-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP),dithioerythritol (DTE), tributylphosphine and dithiothreitol (DTT),advantageously said thiol reducing agent istris(2-carboxyethyl)phosphine or dithiothreitol.

Preferably, the alkylating agent is selected from the group formed byN-ethylmaleimide, iodoacetamide and M-biotin, preferably said alkylatingagent is iodoacetamide (IAA).

Using the parameters of the method according to the present invention,the minimum duration of the enzymatic proteolysis step d), until now alimiting factor, is drastically reduced. Indeed, the minimum duration ofthis enzymatic proteolysis step within the framework of the method ofobtaining peptides according to the present invention is approximately15 minutes (preferably this minimum duration is 5 minutes). Thereduction of the minimum duration required to obtain the peptides at theend of the enzymatic proteolysis step allows reduction, verysignificantly, of the total duration of the method of obtaining peptidesaccording to the present invention, since the latter can be performed inapproximately barely a half-hour.

Generally, the total duration of the method of obtaining peptidesaccording to the invention is less than 1 hour and 45 minutes,preferably less than 1 hour, advantageously less than 45 minutes and,optimally, approximately 30 minutes.

The significant time saving thus obtained is altogether importantparticularly with regard to the requirements inherent in the clinicaland/or pharmaceutical fields. Moreover, the method of obtaining peptidesaccording to the present invention is perfectly suited to complexsamples (plasma, urine, cerebrospinal fluid etc.) which are likely torequire additional fractionation or depletion strategies, in order toisolate the target proteins prior to implementation of the method ofobtaining peptides. Thus, and even including the time necessary forperforming such additional fractionation and depletion strategies, thetreatment time of such complex samples by application of the methodaccording to the invention remains very advantageous in comparison withthe methods of the prior art.

The prior purification treatment of procaryotic and/or eucaryotic cellsamples, before the lysis step a) is known to the man skilled in the artand can in particular implement techniques of centrifugation,filtration, electrophoresis or chromatography. These separativetechniques can be used alone or combined with each other to obtainmultidimensional separation. For example, multidimensionalchromatography can be used by associating separation by ion-exchangechromatography with reverse-phase chromatography, as described by T.Fortin et al. or H. Keshishian et al. In these publications, thechromatographic medium can be in column or in cartridge (solid-phaseextraction). The electrophoretic or chromatographic fraction (or theretention time in mono or multidimensional chromatography) of theproteotypic peptides is characteristic of each peptide and theimplementation of these techniques therefore permits selection of theproteotypic peptide or peptides to be assayed. Such fractionation of thepeptides generated allows an increase in the specificity of thesubsequent assay by mass spectrometry.

An alternative to the techniques of electrophoresis or ofchromatography, for the fractionation of peptides, consists inspecifically purifying N-glycopeptides (and patent application WO2008/066629). Nevertheless, such purification only allows thequantification of the peptides having undergone post-translationalmodification of N-glycosylation type. Now not all the proteins areglycosylated, which therefore limits its use.

In terms of prior purification treatment of the procaryotic and/oreucaryotic cell samples, the method according to the invention,preferably, comprises, before the step a), the following additionalsteps:

-   -   a′) centrifugation of the microorganisms at a rotation speed of        between 3500 and 4500 rpm, preferably of the order of 4000 rpm,        during a time period of between 4 minutes and 6 minutes,        advantageously of 6 minutes, at a temperature of between 15° C.        and 25° C., preferably approximately 20° C.,    -   a″) Elimination of the supernatant and recovery of the pellet        containing the microorganisms,    -   a′″) Adsorption of said pellet in a solvent.

Advantageously, the solvent used within the framework of the presentinvention is selected from a solution of acetonitrile and an aqueoussolution comprising a pH buffer such as carbonate ions, advantageouslysaid solvent is an aqueous solution comprising carbonate ions, such as asolution of ammonium bicarbonate.

As for the step of prior purification treatment of the above-mentionedsample, the method can comprise, where necessary, subsequently to thestep of recovery of the peptides e), steps of concentration of saidpeptides. By way of example, the step of recovery of the peptides e) canbe followed by the following steps:

-   -   e1) Centrifugation with a relative centrifugal force of between        13000 g and 15000 g, advantageously approximately 14000 g, for a        duration of between 25 minutes and 35 minutes, advantageously        approximately 30 minutes, at a temperature of between 2° C. and        6° C., advantageously of approximately 4° C.,    -   e2) recovery of all or part of the supernatant including the        peptides.

According to a first aspect of the present invention, at least the stepsof lysis a) and denaturation b) are performed conjointly/simultaneously.

According to a particular embodiment of this first aspect of theinvention, steps a) and b) are performed conjointly/simultaneously.Thus, and contrarily to the methods of the prior art, the lysis time isused to denature the proteins. This allows shortening of the duration ofthe method as a whole and facilitates its automation.

Within the framework of this particular embodiment, the step ofalkylation of the denatured proteins c) is performed at a temperatureknown to the man skilled in the art to be suitable and able to permitthe performance of the alkylation treatment of said proteins. This stepof alkylation of the proteins c) is preferably performed in the absenceof light and at a temperature of between 10° C. and 60° C., preferablybetween 15° C. and 25° C., advantageously at ambient temperature.

Still within this particular embodiment, the step of enzymaticproteolysis d), for its part, is performed at a suitable temperature,i.e. also known to the man skilled in the art to allow the enzymaticdigestion of the proteins (enzymatic proteolysis). Advantageously, thisstep of proteolysis d) is performed at a temperature of between 30° C.and 60° C., preferably between 37° C. and 55° C., advantageouslyapproximately 50° C.

In this particular embodiment, the duration of the conjoint(simultaneous) steps of lysis a) and of denaturation b) is between 3minutes and 7 minutes, preferably between 4 minutes and 6 minutes,advantageously said duration is approximately 5 minutes.

Moreover, and still within this particular embodiment, the duration ofthe step of alkylation of the denatured proteins c) is also of between 3minutes and 7 minutes, preferably between 4 minutes and 6 minutes,advantageously said duration is approximately 5 minutes.

Still according to this particular embodiment, the method comprises,after the step of alkylation of the denatured proteins c), the followingstep:

-   -   c1) assay of the proteins obtained in said step c),    -   and in which the proteolytic enzyme is added at the step of        enzymatic digestion d) in a ratio by weight (w/w) relative to        the weight of the proteins assayed in step c1) of between 1/5        and 1/15, preferably between 1/8 and 1/12, advantageously        approximately 1/10.

According to a second aspect of the invention, at least steps a)-c) ofthe method according to the invention are performed conjointly(a)+b)+c)).

According to a particular embodiment of this second aspect of theinvention, steps a)-c) (lysis denaturation and alkylation) are performedconjointly (simultaneously).

According to a third aspect of the present invention—particularlypreferred—steps a)-d) are performed conjointly (simultaneously).

According to this third aspect of the present invention—particularlypreferred—steps a)-d) of the method according to the present inventionare performed conjointly (a)+b)+c)+d)).

More precisely, all the steps a)-d) are performed in a same container(for example an eppendorf vial) without requiring the addition ofreagents or manipulation between said steps. This method is thereforeeasily automatable, which represents a major advantage with a view tocost reduction, limiting risks associated with incorrect manipulation,and reducing the volumes to be used (non-limiting list).

Within the framework of the methods according to the second and thirdaspects of the present invention, it is necessary to be sure of thecompatibility of the agents used in the different steps b), c) and d),and more particularly with regard to the compatibility of the denaturingagent and of the alkylating agent, the latter having a natural tendencyto alkylate the denaturing agent introduced in step b).

Preferably, according to these second and third aspects of theinvention:

-   -   the denaturing agent is a thiol reducing agent selected from        tris(2-carboxyethyl)phosphine and dithiothreitol, advantageously        said thiol reducing agent is tris(2-carboxyethyl)phosphine, and        the alkylating agent is iodoacetamide. Indeed, the applicant has        discovered, in surprising manner, that the use of the        TCEP/iodoacetamide (IAA) pair allowed very good yields to be        obtained with regard to the method according to the present        invention.

A further object of the present invention relates to a method ofanalysis of peptides of procaryotic and/or eucaryotic cells comprisingthe following steps:

-   -   i) Obtaining peptides from said procaryotic and/or eucaryotic        cells using the method according to the invention,    -   ii) Analysis of the peptides thus obtained, said analysis being        performed by means of an analysis of mass spectrometry type.

By way of example, this method of analysis of peptides can be, in thefield of microbiology, a method of characterisation of at least onemicroorganism from a sample, comprising, for example, the identificationof said microorganism and the determination of the properties of typing,potential resistance to at least one antimicrobial and virulence factorrelating to said microorganism.

A further example relates to biomarker research, particularly fromcomplex biological samples such as plasma, urine, cerebrospinal fluid,etc.

The present invention also relates to a kit for obtaining peptides fromprocaryotic and/or eucaryotic cells, said kit being specifically suitedto the implementation of the method according to the present invention,said kit comprising:

-   -   a lysis kit allowing lysis to be performed at low concentration        of chaotropic agent(s) of procaryotic and/or eucaryotic cells,        preferably a kit for lysis by sonication,    -   a first solution comprising at least one denaturing agent,        preferably selected from 2-mercaptoethanol,        tris(2-carboxyethyl)phosphine, dithioerythritol,        tributylphosphine and dithiothreitol, advantageously said        denaturing agent is tris(2-carboxyethyl)phosphine or        dithiothreitol,    -   a second solution comprising at least one alkylating agent,        preferably selected from the group formed of any        N-ethylmaleimide, iodoacetamide and M-biotin, preferably said        alkylating agent is iodoacetamide,    -   a third solution comprising at least one proteolytic enzyme such        as a serine protease, preferably selected from the group        consisting in trypsin, chymotrypsin and elastase, advantageously        said proteolytic enzyme is trypsin.

This kit comprises, in optional manner, instructions for use definingits methods of use.

According to a preferred embodiment, said kit comprises:

-   -   a kit for lysis by sonication,    -   a first solution comprising a denaturing agent selected from        2-mercaptoethanol, tris(2-carboxyethyl)phosphine,        dithioerythritol, tributylphosphine and dithiothreitol,        advantageously said denaturing agent is        tris(2-carboxyethyl)phosphine or dithiothreitol,    -   a second solution comprising an alkylating agent consisting in        iodoacetamide,    -   a third solution comprising a proteolytic enzyme consisting in        trypsin.

Preferably, the lysis kit and the first, second and third solutions aresuited and intended to be used conjointly (simultaneously), particularlywithin the framework of the method according to the third aspect of thepresent invention, as indicated above.

Yet another object of the present invention relates to the use of a kitaccording to the invention for implementing the above-mentioned methodof obtaining peptides and/or of the method of analysis of peptides alsomentioned above.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention, its functionality, its applications and its advantageswill be better understood on reading the present description, withreference to the following figures, in which:

FIG. 1 shows diagrammatically a method of preparation of a sample ofmicroorganisms according to the prior art (P0), this method comprisingin particular a step of lysis at high saline concentration (guanidine6M—chaotropic agent) and a long step of tryptic digestion,

FIG. 2 is a diagram showing the different steps of a method according toa first aspect of the present invention (P1),

FIG. 3 shows the data relative to the establishment of a calibrationrange of bovine serum albumin (BSA), thus allowing a correspondence tobe obtained between the optical density (OD) measured at 595 nm and theprotein concentration (mg/ml), within the framework of said first aspectof the present invention (P1)

FIGS. 4, 5 and 6 present the results obtained after implementation ofthe method P1, on each of three microorganisms assayed respectively,i.e. Escherichia coli EC5, Staphylococcus epidermidis SE9 and Candidaalbicans CA16,

FIG. 7 relates to assessment of the possible consequences of the absenceof the step of preheating the ultrasound probe within the framework ofsaid method P1,

FIG. 8 is a diagram showing the different steps of the method accordingto a third, particularly advantageous, aspect of the present invention(P2), in which the denaturing agents used is TCEP (“P2-TCEP”),

FIG. 9 is a graph showing the results of the LC-ESI-MS analysesperformed at the end of the P2-TCEP protocol on each of theabove-mentioned three microorganisms, i.e. Escherichia coli EC5,Staphylococcus epidermidis SE9 and Candida albicans CA16.

FIG. 10 is a diagram showing the different steps of the method accordingto the third, particularly advantageous aspect of the present invention(P2), in which the denaturing agent used is DTT (P2-DTT), —FIG. 11 is agraph showing the results of the LC-ESI-MS analyses performed at the endof the P2-DTT protocol on each of the above-mentioned threemicroorganisms, i.e. Escherichia coli EC5, Staphylococcus epidermidisSE9 and Candida albicans CA16.

DETAILED DESCRIPTION OF THE INVENTION

The examples presented below permit better illustration of the presentinvention. However, these examples must in no case be seen as limitingthe scope of said invention in any manner whatsoever.

Example 1: Protocol P0

Protocol P0 is a protocol for preparation of a sample conventionallyused in the prior art in order to obtain peptides from microorganisms.This method P0 comprises in particular a step of lysis at high saltconcentration (guanidine 6M: chaotropic agent) as well as a long step oftryptic digestion. P0 is used below as a reference in order to evaluatethe quality/efficiency of the protocols for obtaining peptides accordingto a first aspect of the invention (protocol P1) Said protocolP0—described below with reference to FIG. 1—lasts for approximately 24hours and comprises the following steps:

-   -   placing in suspension one to three colonies of a microorganism        101 (such as a bacterium of E. coli type) cultured on a Petri        dish 100 in a vial comprising a solution 111 composed of 100 μl        of guanidine hydrochloride 6M, 50 mM Tris-HCl, pH 8, and leaving        the lysis reaction 110 to take place for a period of        approximately 10 minutes;    -   introducing, at the denaturation/reduction step 120, a solution        121 comprising 3.5 μl of DTT at a concentration of 150 mM, so as        to obtain a final 5 mM solution of DTT; this step of        denaturation/reduction being performed in approximately 30        minutes;    -   then performing the alkylation step 130 by introducing a        solution 131 of IAA at a concentration of 150 mM and leaving the        alkylation reaction 130 to take place for approximately 45        minutes so as to perform locking (protection) of the thiol        functions on the denatured proteins;    -   prior to the step of tryptic digestion 150, performing a step of        dilution 140 by adding 500 μl of a solution 141 comprising 50 mM        of NH₄HCO₃, in order to diminish the high concentration of        chaotropic agent (guanidine 6M) in order not to impair the        enzymatic activity of the trypsin at the subsequent step of        tryptic digestion 150;    -   performing this tryptic digestion step 150 by introducing 0.5 to        3 μl of a trypsin solution 151 measured at 1 μg/μL; leaving the        enzymatic digestion to take place for 6 to 20 hours (duration        allowing the peptides 153 to be obtained);    -   stopping in step 160 said tryptic digestion 150 by adding 2 μl        of formic acid 161; then    -   separating the peptides 171 thus obtained, for example by        centrifugation to eliminate the non-soluble species which could        hamper the subsequent analytical steps and sampling them in        order to perform, where necessary, subsequent steps of analysis        (analyses).

Example 2: Protocol P1

A method according to a first aspect of the invention—called “protocolP1” or “method P1”—is described below, with reference to FIG. 2.

This protocol P1 permits very rapid preparation of the sample prior tothe subsequent analysis steps, performed, for example, by massspectrometry. The duration of all of the steps of protocol P1 isapproximately 30 minutes. It comprises in particular the followingessential steps:

-   -   lysis and reduction 208 (performed conjointly/simultaneously),        in the presence of DTT 5 min under ultrasound,    -   alkylation 212: in the presence of IAA, 5 min at ambient        temperature and without agitation,        and        enzymatic proteolysis (also called enzymatic digestion) 214: in        the presence of trypsin, 15 min at 50° C.

As indicated above, the protocol P1 is performed in approximately 30minutes and gives similar results (MRM analyses) to those obtained byprotocol P0 (cf. Example 1—duration of treatment: 24 h), on each of thethree microorganisms assayed, i.e.: Escherichia coli EC5 (Gram-;hereinafter designated “EC5”), Staphylococcus epidermidis SE9 (Gram+;hereinafter designated “SE9”) and Candida albicans CA16 (yeast;hereinafter designated “CA16”).

Equipment and Method

2.1. Products Used

-   -   BSA/Sigma/reference A9085-5G/lot No.: 097K1513    -   Bradford reagent: “Quick Start Bradford Dye Reagent        1X”/Bio-Rad/reference 500-0205/control 210006065    -   Formic acid/Fluka/reference: 06450    -   Iodoacetamide, (IAA)/Sigma/I6125-5G/lot 099K5300/MM=184.96    -   DL-1.4-Dithiothreitol 99%,        (DTT)/Acros/165680010/lotA0269816/MM=154.24    -   Ammonium bicarbonate/Sigma/A6141-500 g/lot No. 117K0039/MM=79.06    -   Ammonium hydroxide, NH₄OH/28% ammonia/reference:        21190292/MM=17.03 g    -   Trypsin/Promega/reference V511 (storage at −20° C.)    -   “Suspension medium” (sterile water) bioMérieux (ref.: 70640)    -   Escherichia coli strain, ATCC No.: 11775T    -   Staphylococcus epidermidis strain, ATCC No.: 14990    -   Candida albicans strain, ATCC No.: 18804

2.2. Preparation of the Buffer Solutions

-   -   Buffer No. 4: 50 mM ammonium bicarbonate pH8/storage 1 month at        +4° C.    -   For 50 ml buffer: 197.6 mg of bicarbonate qsp 50 ml H₂O/pH=7.9;        add approximately 10 μl of NH₄OH to obtain pH=8    -   Buffer No. 5: 150 mM DTT/to be prepared extemporaneously    -   For 1 ml of buffer: 23.1 mg of DTT qsp 1 ml bicarbonate buffer        No. 4    -   Buffer No. 6: 150 mM IAA/to be prepared extemporaneously    -   For 1 ml buffer: 27.7 mg of IAA qsp 1 ml bicarbonate buffer No.        4

2.3. Equipment

-   -   Centrifuge “APPLI 24”/Prolabo    -   1.5 ml vials “Safe Lock” Eppendorf, ref: 0030120.086    -   Centrifuge “benchtop”/Eppendorf/ref 5415C    -   Thermomixer “comfort”/Eppendorf    -   Microplate reader (595 nm)+microplates+modules    -   Spectrophotometer UVIKON+80 μl quartz cuvettes    -   Culture dishes BMX: COS (ref.: 43041) and SDA (ref.: 43555)    -   Densichek Plus BioMérieux: reference: 21250    -   Ultrasound probe “Hielscher”; ref: PN-66-NNN    -   Lysis beads 0.1 mm (small diameter): “Zirconia/Silica        beads”/Roth/N033.1    -   Lysis beads 1 mm (large diameter): “Silibeads typ 1/1.3 mm/ref:        4504/VWR    -   Bridges/Dutscher/reference: 011870A

2.4. Protocol

2.4.1. Steps 200, 202, 204, 206, 208 and 210

Firstly, the buffer solutions No. 5 (150 mM DTT) and No. 6 (150 mM IAA150 mM) are prepared.

Using a “spoon” spatula, 50 mg of 0.1 mm diameter beads (small diameterbeads) and 50 mg of 1 mm diameter beads (large diameter beads) areweighed and are introduced into the No. 1 vial. The mixture of beads ofsmall diameter and large diameter is represented in FIG. 2 by thenumerical reference 2061.

One to three colonies 110 are taken from a Petri dish 100 and are placedin suspension in water in step 200. The concentration of bacteria in thesuspension thus obtained is estimated by conventional turbiditymeasurement methods (measurement of absorption at 550 nm). A volume ofsuspension corresponding to 1·10⁸ CFU is taken and then centrifuged, instep 202, at 4000 rpm for 5 min, at ambient temperature. At the end ofthis centrifugation step 202, the pellet is adsorbed (EC5, CA16 and SE9)in step 204 in 100 μl of the No. 4 buffer solution (vial No. 2), then3.5 μl of 150 mM DTT (buffer No. 5) are added, still in step 204, toobtain a final DTT concentration of 5 mM in vial No. 2. Vortexing isapplied for 2 seconds (maximum power) to homogenise the contents of thisvial No. 2 and the mixture is pipetted in order to transfer it into vialNo. 1, in step 206, containing the mixture of beads of “small diameter”and “large diameter” 2061. Vial No. 2 is eliminated.

A bridge is placed on vial No. 1 to prevent it from opening during lysisby sonication.

Vial No. 1 is then introduced into one of the orifices of the HielscherUltrasound probe (the 6 orifices at the end of the probe are supposed tobe identical according to the supplier), and then 5 min are timed toperform lysis/reduction 208 (settings Amplitude 100/Cycle 1); thetemperature of the mixture, in the vial, reaches approximately 95° C.

Vial No. 1 is removed from the orifice of the probe by means of a“lever” holding the vial by the bridge, then this vial No. 1 is cooled,in step 210, by storing it for 1 minute in ice in order to return thetemperature of the mixture in the vial to ambient temperature.

This vial No. 1 is then briefly centrifuged by means of a benchtopcentrifuge (at the end of step 210), in order to recover the liquidpresent in the cap and on the walls.

2.4.2. Alkylation Step 212 (Locking of the Disulphide Bridges of theProteins by Methylation with IAA)

9 μl of solution of 150 mM IAA (buffer No. 6) are added into vial No. 1in step 212 to obtain a final molarity of 12.5 mM, said vial No. 1 thenbeing vortexed 2 seconds (maximum power) for homogenisation.

The alkylation reaction 212 is left to take place for 5 min at ambienttemperature, in the absence of light.

At this stage, two options are possible, i.e.:

-   -   protocol P1 is followed with the step of tryptic digestion 214        (cf. section 2.4.4. below), using a predefined quantity of        trypsin, or    -   before this step of tryptic digestion 214 (also called tryptic        proteolysis) is performed an assay of the proteins 213 (cf.        2.4.3. below), to be executed in a vial No. 3 which is used only        to implement the assay of the proteins 213, in order to evaluate        the quantity of trypsin to be added at the step of tryptic        digestion 214 as a function of the protein concentration assayed        at the end of the alkylation step 212.

2.4.3. Protein Assay 213 (According to the Bradford Method)

The protein assay is performed on the supernatant of the lysatecentrifuged for 5 minutes at 14000 rpm. It is therefore necessary, in afirst stage, to vortex vial No. 1 (homogenisation of the lysate), andthen to pipette the lysate from said vial No. 1 (a part of the 0.1 mmbeads is often recovered), to transfer this lysate into a vial (vial No.3) and to centrifuge this vial No. 3 at 14000 rpm for 5 minutes. Theassay is then performed on the supernatant. The vial No. 3 comprisingthe remaining beads is eliminated.

The quantity of proteins is evaluated as a function of a calibrationrange of BSA diluted in carbonate buffer and assayed in parallel withthe centrifuged lysate (range established from a 1 mg/ml BSA solutionand then preparation of 0.1/0.2/0.3/0.4/0.6/0.8 and 1 mg/ml solutions).

The assay is performed on 4 μl of the supernatant of the centrifugedlysate and 4 μl of each of the solutions of the range, in 200 μl ofBradford reagent, on microplate, with the optical density (OD) read at595 nm.

An Example of calibration range is shown in FIG. 3.

2.4.4. Tryptic Digestion of the Proteins 214

The thermomixer is preheated, in advance, for 15 minutes at 50° C.

A solution of trypsin adsorbed extemporaneously is used: 20 μg flask ofPromega trypsin in 20 μl of the adsorption solution contained in the kit(final concentration 1 μg/μL).

As is known to the man skilled in the art, the maximum activity oftrypsin occurs at a pH of 7/9.

If a protein assay has been performed in step 213, the 1 μg/μ1 trypsinis added in step 214 with a protease/protein ratio by weight (w/w) of1/10, or 1 (=1 μg) for 10 μg of proteins.

Conversely, if such a protein assay 213 has not been performed betweenthe alkylation step 212 and that of tryptic digestion 214, the 1 μg/μ1trypsin is introduced into vial No. 1, in step 214, at a standardquantity of 10 μL. Indeed the Applicant has determined that thisstandard quantity of trypsin allowed satisfactory tryptic digestion tobe obtained under all the pertinent conditions of quantities ofmicroorganisms with regard to the desired peptide analysis applications.

Still in step 214, and subsequently to the addition of trypsin, themixture is vortexed for 2 seconds (maximum power) for homogenisation.

This mixture is then incubated in the thermomixer, 15 minutes at 50° C.,850 rpm.

2.4.5. Stopping the Tryptic Digestion 216

Stopping of the tryptic digestion is effected by an addition of formicacid (qsp pH below 4) in vial No. 1 in step 216. Indeed, trypsin becomesinactive at a pH lower than 4 (this phenomenon is reversible and trypsinbecomes active again at a pH greater than or equal to 4).

The pH of the sample was approximately 8 (pH verified with the pHmeter/microtube special probe) before addition of 0.5 μl of formic acid.It is approximately 3 after addition of the latter in step 216. Themixture is then vortexed for 2 seconds (maximum power) forhomogenisation.

2.4.6. Steps 218, 220, 222 and 224

The peptides resulting from the tryptic digestion are in thesupernatant.

The supernatant of vial No. 1 is pipetted (a part of the 0.1 mm beads isoften recovered) and transferred into another vial (vial No. 4) in step218 and then centrifuged, in step 220, for 30 minutes at 15000 g, at atemperature of 4° C.

85 μl of supernatant including the peptides are then recovered in step222 and introduced into another vial (vial No. 5) in step 224. Thelatter vial is stored in the freezer at −20° C.

The peptides contained in vial No. 5 are then analysed by MRM in orderto determine the quality/efficiency of the protocol P1 (step ofvalidation of the results). The results of these analyses are presentedbelow.

2.5. Validation of the Results

As indicated above, this protocol for obtaining peptides P1 wasvalidated by MRM. The results, for each of the three microorganismsstudied (EC5, SE9 and CA 16), respectively, are presented in FIGS. 4, 5and 6.

FIG. 4 shows the results of analysis in MRM mode of peptides obtainedwith the protocol P1 from 1^(e)8 CFU of E. coli EC5. The experiment wasreproduced three times (columns EC1, EC2, EC3) and compared with theresults obtained from the same inoculum using the protocol P0 (hatchedbar). The bars represent the sum of the areas under the peaks of thecorrectly detectable peptides in the mass chromatogram obtained byLC-ESI-MS analysis in MRM mode. This total area represents the intensityof the signals obtained and is linked to the concentration of thecorresponding peptides in the analysed solution. The greater this area,the higher is this concentration. The figures also indicate (pointsconnected by lines) the number of correctly detected peptides, out of apossible total of 60 for the analysis settings which have been selected.

The first graph (upper part of FIG. 4) shows the data obtained bystandardising the quantity of proteins present in the sample analysed byLC-ESI-MS. The second graph (lower part of FIG. 4) shows the dataobtained by modifying the volume of reaction product P1 so as to analysethe equivalent of 1e7 initial CFU. In this case, the comparison of theareas under the peaks permits comparison of the overall yields obtainedfrom the different samples.

In conclusion it appears that:

-   -   the method P1 is reproducible for the sum of the accumulated        areas under the peaks of the detected peptides, which means that        the intensities of these peaks are reproducible overall and        suggests that the yields of the different steps of the protocol        are reproducible,    -   this method is equivalent to the reference method P0 (presented        in Example 1) in terms of the number of peptides detected with        regard to EC5,    -   this method P1 permits more efficient digestion by trypsin than        under the conditions of the protocol P0, as witnessed by the        fact that the analysis of equivalent protein masses before        digestion results in greater peptide signal intensities for P1        than for P0.    -   conversely, with an equal quantity of bacteria at the beginning        of the protocol, the peptide detection intensities are        equivalent for the two protocols, which would seem to suggest        that the pre-digestion steps of the protocol P1 are less        efficient than in the protocol P0.

FIG. 5 is the equivalent of the first graph of FIG. 4 (in the upperpart) in the case in which the microorganism studied is S. epidermidisSE9. According to this FIG. 5, it appears that the conclusions drawnabove in the case of E. coli EC5 are also valid in the case of otherspecies of bacteria—in particular S. epidermidis SE9. This is especiallytrue as the equivalent mass of proteins injected is smaller under the P0conditions than under the P1 conditions.

In conclusion, and despite the fact that the injected quantity issmaller for P0, the method P1 can be regarded as at least equivalent toP0 regarding the detection of peptides of SE9.

FIG. 6, for its part, shows that regarding the detection of peptides ofCA16:

-   -   the method P1 is reproducible for the sum of the areas and,    -   this method is superior to the reference method P0.

2.6. Influence of the Step of Preheating the Ultrasound Probe

The protocol P1 detailed above comprises a step of preheating the“Hielscher” ultrasound probe by allowing this probe to operate “empty”under the conditions then used for the lysis step of the protocol P1 for1 hour. Under these conditions, the temperature of the vibrating blockof the probe is approximately 95° C. This preheating step beingsusceptible to reducing the lifetime of the ultrasound probe, theapplicant has sought to determine the possible consequences of anomission of said step of preheating this ultrasound probe within theframework of the protocol P1. The protocol P1 thus modified is calledP1′.

FIG. 7 shows the quantities of assayed proteins after the step of lysisby sonication and reduction 208 (cf. FIG. 2) after different operatingtimes of the sonication probe (0, 20, 40, 60, 80 minutes, the time 0 mincorresponding to the use of a probe used after a time of sufficientinactivity for the vibrating block to be at ambient temperature). Theprotein concentrations have been determined by a Bradford test and areshown on the graphs and expressed in μg for 1e8 bacterial cells.

This assay can be conducted by the Bradford method or by any othersuitable method known to the man skilled in the art.

The results shown in this FIG. 7 confirm that it is not necessary topreheat the probe before its use to obtain high-quality bacterial lysis.

Moreover, the MRM analyses also confirm that the tryptic digestiontrials of the three strains EC5, SE9 and CA16, without preheating of theultrasound probe used to perform the lysis (protocol P1′), give resultssuperior to the reference protocol P0.

Besides the advantages inherent in the methods for obtaining peptidesaccording to the present invention (cf. above), these methods permitdispensing with the prior step of preheating of the ultrasound probe,within the framework of lysis by sonication. This results in particularin an increased longevity of said ultrasound probe as well as in anenergy saving.

Example 3: Protocol P2

A third aspect of the invention, called “protocol P2” (or “method P2”),is described below, with reference to FIG. 8. This method P2 isparticularly preferred within the context of the present invention.

In this Example 3, the method P2 is used to obtain peptides frommicroorganisms.

This method P2 is a protocol for very rapidly obtaining peptides fromprocaryotic and/or eucaryotic cells, which can be used in particularprior to steps of analysis by mass spectrometry type.

Just like the protocol P1, this protocol P2 is also performed inapproximately 30 min but combines the steps of lysis,denaturation/reduction, alkylation and enzymatic digestion in a singlestep 810. These different steps are indeed performedconjointly/simultaneously in a single and same vial at 50° C., underultrasound for 30 minutes. Thus, there is no need to perform manualinterventions between these steps, nor to add reagents during themethod.

The protocol P2 is therefore easy to automate, avoids possible errors ofmanipulation and permits the use of smaller volumes. Moreover, it alsoallows the analysis of complex samples (urine, plasma, cerebrospinalfluid), such as explained above.

This protocol P2 gives similar results (LC-ESI-MS analyses) to theresults of the protocol P1 described in example 2, for two of the threemicroorganisms assayed: Escherichia coli (Gram-) and Staphylococcusepidermidis (Gram+) both in number of peptides and in cumulated area.

Equipment and Method

3.1 Products Used

-   -   Formic acid/Fluka/reference: 06450    -   Iodoacetamide, (IAA)/Sigma/16125-5G/MM=184.96    -   Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride 0.5 M        solution/Sigma/646547    -   Ammonium bicarbonate/Sigma/A6141-500 g/MM=79.06    -   Ammonium hydroxide, NH₄OH/28% ammonia/reference:        21190292/MM=17.03 g    -   Trypsin/Promega/reference V511 (storage at −20° C.)    -   “Suspension medium” (sterile water) bioMérieux (ref.: 70640)    -   Escherichia coli strain ATCC No.: 11775T    -   Staphylococcus epidermidis strain ATCC No.: 14990    -   Candida albicans strain, ATCC No.: 18804

3.2 Preparation of the Buffer Solutions

-   -   Buffer No. 4: 50 mM ammonium bicarbonate pH8/storage 1 month at        +4° C.    -   For 50 ml buffer: 197.6 mg of bicarbonate qsp 50 ml H₂O/pH=7.9;        approximately 10 μl addition of NH₄OH to obtain pH=8    -   Buffer No. 5′: 150 mM TCEP/to be prepared        extemporaneously/dilution in buffer no. 4    -   Buffer No. 6: 150 mM IAA/to be prepared extemporaneously    -   For 1 ml buffer: 27.7 mg of IAA qsp 1 ml bicarbonate buffer No.        4

3.3 Equipment

-   -   Centrifuge “APPLI 24”/Prolabo    -   1.5 ml vials; “Safe Lock” Eppendorf, ef 0030120.086    -   Centrifuge “benchtop”/Eppendorf/ref 5415C    -   Spectrophotometer UVIKON+80 μl quartz cuvettes    -   BMX culture dishes BMX: COS (ref.: 43041) and SDA (ref.: 43555)    -   Ultrasound probe “Hielscher”; ref: PN-66-NNN    -   0.1 mm lysis beads (small diameter): “Zirconia/Silica        beads”/Roth/N033.1    -   1 mm lysis beads (large diameter): “Silibeads typ 1/1.3 mm; ref:        4504/VWR    -   Bridges/Dutscher/reference: 011870A

3.4 Protocol

3.4.1. Preliminary Steps 800, 802, 804 and 806

In practice, firstly buffer solutions No. 5′ (dilution of a solution of500 mM TCEP to 150 mM in bicarbonate buffer No. 4) and No. 6 (27.7 mg/mlof IAA in bicarbonate buffer No. 4) are prepared.

Then, using a “spoon” spatula, 50 mg of beads of 0.1 mm diameter and 50mg of beads of 1 mm diameter are weighed and are introduced into a vialwith a capacity of 1.5 ml. As indicated above, the mixture of the beadsof “small diameter” and of “large diameter” is designated by thenumerical reference 8061, still in FIG. 8.

One to three colonies 110 are taken from a Petri dish 100, which areplaced in suspension in water in step 800. The concentration of bacteriain the suspension thus obtained is estimated by conventional turbiditymeasurement methods (measurement of absorption at 550 nm). A volume ofsuspension corresponding to 1.108 CFU is taken and then centrifuged instep 802, at 4000 rpm for 5 minutes at ambient temperature. At the endof this centrifugation step 802, the pellet is adsorbed (EC5, CA16 andSE9) in step 804 in 100 μl of No. 4 buffer solution vial No. 2) andthen, still in step 804, are added 3.5 μl of 150 mM TCEP (buffer No. 5′)to obtain a final TCEP concentration of 5 mM in vial No. 2. Vortexing isapplied for 2 seconds (maximum power) to homogenise the contents of thisvial No. 2 and this mixture is pipetted in order to transfer it, in step806, into vial No. 1 containing the 8061 beads. Vial No. 2 is theneliminated.

3.4.2. Steps 808, 810 and 812

Then are introduced into vial No. 1, in step 808:

-   -   9 μl of solution of 150 mM IAA (buffer No. 6) in order to obtain        a final molarity of 12.5 mM; and    -   10 μl of 1 μg/μl trypsin.

Following this, vortexing is applied for 2 seconds (maximum power) forhomogenisation.

A bridge is placed on vial No. 1 to prevent it from opening during lysisby sonication.

This vial No. 1 is then introduced into one of the orifices of theHielscher probe (the 6 orifices at the end of the probe are supposed tobe identical according to the supplier), and then 30 minutes are timed(settings Amplitude 100/Cycle 0.5) in order to allow the reactions oflysis, of reduction, of alkylation and of tryptic digestion to takeplace conjointly/simultaneously. The temperature of the mixture in vialNo. 1, at step 810, reaches approximately 50° C.

Vial No. 1 is then removed from the orifice of the probe by means of a“lever” holding the vial by the bridge, and then this vial No. 1 iscooled, in step 812, by storing it for 1 minute in ice in order toreturn the temperature of the mixture in the vial to ambienttemperature.

Summarising, the main step 810 allows the conjoint/simultaneousoccurrence of:

-   -   a. Lysis of the bacteria/reduction of the proteins (TCEP breaks        the disulphide bridges): obtaining of the denatured/reduced        proteins    -   b. Alkylation: step of locking the disulphide bridges of the        proteins by methylation with IAA    -   c. Tryptic digestion of the proteins: obtaining the peptides.

3.4.3. Stopping the Tryptic Digestion 814

Stopping the tryptic digestion is performed by addition of formic acid(qsp pH below 4) in vial No. 1, in step 814. As indicated above, thefact of lowering the pH below 4 reversibly inactivates the enzymaticactivity of trypsin.

The pH of the sample is approximately 8 (pH verified with the pHmeter/microtube special probe) before the addition of 0.5 μl of formicacid. It is approximately 3 after addition of the latter in step 814.The mixture is then vortexed for 2 seconds (maximum power) forhomogenisation.

3.4.4. Steps 816, 818, 820 and 822

The peptides obtained at the end of the main step 810 are in thesupernatant. The latter is therefore pipetted (a part of the 0.1 mmbeads is often recovered) and transferred, in step 816, into anothervial (vial No. 3), and then centrifuged, in step 818, for 30 minutes, at15000 g and at 4° C. 90 μl of supernatant comprising the peptides arethen recovered in step 820 and introduced into another vial (vial No. 4)in step 822. The latter is stored in the freezer at a temperature of−20° C.

3.5. Validation of the Results

The peptides contained in vial No. 4 are then analysed by LC-ESI-MS inorder to determine the quality/efficiency of lysis protocol P2 (step ofvalidation of the results). The results, for each of the threemicroorganisms studied, are presented in FIG. 9. Said protocol P2 isdesignated “P2-TCEP” in this FIG. 9, with reference to the TCEP, used asa denaturing agent in the present example 3.

As explained above, the values shown in this FIG. 9 correspond to thenumbers of the correctly detected targeted peptides as well as to thecumulated area under the peak of these peptides in the mass spectrumobtained by LC-ESI-MS in MRM mode.

As shown in this FIG. 9, this protocol P2 produces similar results tothose obtained by the above-mentioned protocol P1 for two of the threemicroorganisms assayed, i.e. Escherichia coli and Staphylococcusepidermidis. The results obtained for Candida albicans are satisfactoryfor the number of peptides.

As indicated above, the methods (protocols) of obtaining peptidesaccording to the present invention fall within the framework of thepreparation of samples of procaryotic and/or eucaryotic cells for theirsubsequent analysis.

Example 4: Protocol P2 with DTT (“P2-DTT”)

The protocol P2 the object of example 3 above was reproduced replacingthe TCEP with another denaturing agent, i.e. DTT. For purposes ofclarity, this method P2 with DTT is called “P2-DTT”.

The protocol P2-DTT is described below, with reference to FIG. 10.

The protocol P2-DTT gives similar results (LC-ESI-MS analyses) to theprotocol P1 described in example 2, for two of the three microorganismsassayed: Escherichia coli (Gram-) and Staphylococcus epidermidis (Gram+)both in number of peptides and in cumulated area.

Equipment and Method

4.1 Products Used

-   -   Formic acid/Fluka/reference: 06450    -   Iodoacetamide, (IAA)/Sigma/16125-5G/MM=184.96    -   DL-1,4-Dithiothreitol 99%,    -   (DTT)/Acros/165680010/lotA0269816/MM=154.24    -   Ammonium bicarbonate/Sigma/A6141-500 g/MM=79.06    -   Ammonium hydroxide, NH₄OH/28% ammonia/reference:        21190292/MM=17.03 g    -   Trypsin/Sigma/T0303 (storage at −20° C.)    -   “Suspension medium” (sterile water) bioMérieux (ref.: 70640)    -   Escherichia coli strain, ATCC No.: 11775T    -   Staphylococcus epidermidis strain, ATCC No.: 14990    -   Candida albicans strain, ATCC No.: 18804

4.2 Preparation of the Buffer Solutions

-   -   Buffer No. 4; 50 mM ammonium bicarbonate pH8/storage 1 month at        +4° C.    -   For 50 ml buffer: 197.6 mg of bicarbonate qsp 50 ml H2O/pH=7.9;        addition of approximately 10 μl of NH₄OH to obtain pH=8    -   Buffer No. 5: 150 mM DTT/to be prepared extemporaneously    -   For 1 ml of buffer: 23.1 mg of DTT qsp 1 ml bicarbonate buffer        No. 4    -   Buffer No. 6: 150 mM IAA 150 mM/to be prepared extemporaneously    -   For 1 ml buffer: 27.7 mg of IAA qsp 1 ml bicarbonate buffer No.        4

4.3 Equipment

-   -   Centrifuge “APPLI 24”/Prolabo    -   1.5 ml vials; “Safe Lock” Eppendorf, ref: 0030120.086    -   Centrifuge “benchtop”/Eppendorf/ref 5415C    -   Spectrophotometer UVIKON+80 μl quartz cuvettes    -   BMX culture dishes: COS (ref.: 43041) and SDA (ref.: 43555)    -   Ultrasound probe “Hielscher”; ref. PN-66-NNN    -   0.1 mm lysis beads (small diameter): “Zirconia/Silica        beads”/Roth/N033.1    -   1 mm lysis beads (large diameter): “Silibeads typ 1/1.3 mm/ref:        4504/VWR    -   Bridges/Dutscher/reference: 011870A

4.4 Protocol

4.4.1. Preliminary Steps 1000, 1002, 1004 and 1006

Firstly, the buffer solutions No. 5 (150 mM DTT) and No. 6 (150 mM IAA)are prepared.

Then, using a “spoon” spatula, 50 mg of 0.1 mm diameter beads and 50 mgof 1 mm diameter beads are weighed and are introduced into a vial with acapacity of 1.5 ml. As indicated above, the mixture of the beads of“small diameter” and of “large diameter” is designated by the numericalreference 10061, still in FIG. 10.

One to three colonies are taken 110 from a petri dish 100, and areplaced in suspension in water in step 1000. The concentration ofbacteria in the suspension thus obtained is estimated by conventionalmethods of turbidity measurement (measurement of absorption at 550 nm).A volume of suspension corresponding to 1.10⁸ CFU is taken and thencentrifuged in step 1002, at 4000 rpm for 5 minutes at ambienttemperature. At the end of this step of centrifugation 1002, the pelletis adsorbed (EC5, CA16 and SE9) in step 1004 in 100 μl of the buffersolution No. 4 (Vial No. 2), then, still in step 1004, are added 3.5 μlof 150 mM DTT (buffer No. 5) to obtain a final DTT concentration of 5 mMin vial No. 2. Vortexing is applied for 2 seconds (maximum power) tohomogenise the contents of this vial No. 2 and this mixture is pipettedin order to transfer it, in step 1006, into vial No. 1 containing the10061 beads. Vial No. 2 is then eliminated.

4.4.2. Steps 1008, 1010 and 1012

Into vial No. 1, in step 1008, are then introduced:

-   -   9 μl of 150 mM solution of IAA (buffer No. 6) in order to obtain        a final molarity of 12.5 mM; and    -   10 μl of 1 μg/μl trypsin.

After that, vortexing is applied for 2 seconds (maximum power) forhomogenisation. A bridge is placed on vial No. 1 to prevent it fromopening during lysis by sonication.

This vial No. 1 is then introduced into one of the orifices of theHielscher probe (the 6 orifices at the end of the probe are supposed tobe identical according to the supplier), and then 30 minutes are timed(settings Amplitude 100/Cycle 0.5) in order to allow the reactions oflysis, of reduction, of alkylation and of tryptic digestion to takeplace conjointly/simultaneously. The temperature of the mixture in vialNo. 1, at step 1010, reaches approximately 50° C.

Vial No. 1 is then removed from the orifice of the probe by means of a“lever” holding the vial by the bridge, and then this vial No. 1 iscooled, in step 1012, by storing it for 1 minute in ice in order toreturn the temperature of the mixture of the vial to ambienttemperature.

Summarising, the main step 1010 allows taking placeconjointly/simultaneously of:

-   -   a. Lysis of the bacteria/reduction of the proteins (DTT breaks        the disulphide bridges): obtaining of the denatured/reduced        proteins    -   b. Alkylation: step of locking the disulphide bridges of the        proteins by methylation with IAA    -   c. Tryptic digestion of the proteins: obtaining of the peptides.

4.4.3. Stopping the Tryptic Digestion 1014

Stopping the tryptic digestion is performed by addition of formic acid(qsp pH lower than 4) in vial No. 1, in step 1014. As indicated above,the fact of lowering the pH below 4 reversibly disables the enzymaticactivity of trypsin.

The pH of the sample is approximately 8 (pH verified with the pHmeter/microtube special probe) before the addition of 0.5 μl of formicacid. It is approximately 3 after addition of the latter in step 1014.The mixture is then vortexed for 2 seconds (maximum power) forhomogenisation.

4.4.4. Steps 1016, 1018, 1020 and 1022

The peptides obtained at the end of the main step 1010 are in thesupernatant. The latter is therefore pipetted (a part of the 0.1 mmbeads is often recovered) and transferred, in step 1016, into anothervial (vial No. 3), and then centrifuged, in step 1018, for 30 minutes,at 15000 g and at 4° C.

90 μl of supernatant comprising the peptides are then recovered in step1020 and introduced into another vial (vial No. 4) in step 1022. Thelatter is stored in the freezer at a temperature of −20° C.

4.5. Validation of the Results

The peptides contained in vial No. 4 are then analysed by LC-ESI-MS inorder to determine the quality/efficiency of the lysis protocol P2-DTT(step of validation of the results). The results, for each of the threemicroorganisms studied, are presented in FIG. 11.

As explained above, the values shown in this FIG. 11 correspond to thenumbers of targeted peptides correctly detected as well as to thecumulated area under the peak of these peptides in the mass spectrumobtained by LC-ESI-MS in MRM mode.

As shown in this FIG. 11, this protocol P2 with DTT produces resultssimilar to those obtained by the protocol P1 mentioned above for two ofthe three microorganisms assayed, i.e. Escherichia coli andStaphylococcus epidermidis. The results obtained for Candida albicansare satisfactory for the number of peptides.

Moreover, it proves that the use of DTT instead of TCEP as denaturingagent has no apparent impact either on the number of peptides or on theoverall cumulated area.

As indicated above, the methods (protocols) of obtaining peptidesaccording to the present invention fall within the framework of thepreparation of samples of procaryotic and/or eucaryotic cells for theirsubsequent analysis.

The analysis of these samples can in particular allow biomarker researchand can be applied, inter alia, to the analysis of complex biologicalsamples such as plasma, urine, cerebrospinal fluid, etc.

In the field of microbiology, the peptides obtained by the methodaccording to the invention can allow the characterisation of at leastone microorganism from a sample, comprising for example, theidentification of said microorganism and the determination of theproperties of typing, potential resistance to at least one antimicrobialand virulence factor relating to said microorganism.

This method of characterisation of at least one microorganism can proveparticularly useful in the medical, pharmaceutical or agro-food field.

The determination of the properties of typing, resistance to at leastone antimicrobial and virulence factor is performed by mass spectrometryusing proteins, peptides and/or metabolics as markers of said propertiesof typing, resistance to at least one antimicrobial and virulencefactor. Preferably the mass spectrometry is of MS/MS type,advantageously this mass spectrometry is MRM.

Preferably, the determination of the products of typing, resistance toat least one antimicrobial and virulence factor is performed in the samemass spectrometry apparatus simultaneously.

Optimally, mass spectrometry of MS/MS type, and advantageously of MRMtype, can be envisaged as the step of confirmation of to confirm theidentification of the microorganism.

Indeed, the characterisation of the microorganisms is fundamental bothin the clinical field and in the industrial field. Thus, for example,the identification of resistance to antimicrobials such as antibiotics,and the detection of virulence factors are essential elements forensuring optimal treatment of patients. Similarly, typing is crucial forepidemiological studies and for combating nosocomial illnesses.

By typing of a microorganism, is understood the differentiation ofseveral strains within a same species. Typing has an epidemiologicalvalue, the clinician knows whether the strain isolated in the patientcomes from the same source as other apparently identical strainsisolated in other patients or in the environment. This thus allows thesource of infection to be revealed within a hospital or in the case offood poisoning. As non-limiting examples of markers of properties oftyping in bacteria, can be cited peptides presenting characteristicmutations such as the transcription products of the genes adk, fumC,gyrB, icd, mdh, purA and recA of Escherichia coli, and those of thegenes arc, aroE, glpF, gmk, pta, tpi and yQiL of Staphylococcus aureus.As non-limiting examples of markers of typing properties in theprotozoa, can be cited the products of the chitinase gene of Entamoebahistolytica and E. dispar. As non-limiting examples of markers of typingproperties in the viruses, can be cited the products of the polymerasegene of the human immunodeficiency virus. Lastly, as non-limitingexamples of markers of typing properties in yeasts, can be cited thetranscription products of the gene fragments aat1a, acc1, adp1, mpib,sya1, vps13, and zwf1b of Candida albicans.

By determination of the resistance to at least one antimicrobial, isunderstood the determination of the susceptibility of a microorganism tobe destroyed by an antimicrobial. Thus, if the microorganism is abacterium, the antimicrobial against which it can develop a resistanceis an antibiotic, if it is a protozoan, the antimicrobial is anantiparasitic, if it is a virus, the antimicrobial is an anti-viral,and, if it is a yeast, the antimicrobial is an antifungal The proteinsinvolved in the resistance mechanisms will differ depending on thefamily and the species. As non-limiting examples of markers ofresistance to at least one antibiotic useful with bacteria, can be citedthe transcription products of the MecA gene of Staphylococcus aureus,conferring resistance to Methicillin, and permitting indication ofwhether the strains are methicillin-resistant (MRSA strains) ormethicillin-sensitive (MSSA strains). The protein TEM-2 can also becited which permits indication of whether the strains of Escherichiacoli are resistant to the penicillins but sensitive to other classes ofantibiotics of the cephalosporin or carbapenem type. Another marker isthe enzyme called KPC (for Klebsiella Pneumoniae Carbapenemase) whichconfers resistance to the carbapenems. Another example of a resistancemarker for Staphylococcus aureus is the metabolic profile representativeof resistance to vancomycin such as described by Alexander E. et al inthe poster “Metabolomics-based approach to antibiotic resistance inStaphylococcus aureus” presented to the ASMS congress, 2009. As anon-limiting example of markers of resistance to at least oneanti-parasitic useful with protozoans, can be cited dismutase superoxidecontaining iron (Fe-SOD) and peroxiredoxin increased expression of whichconfers resistance to metronidazole. As a non-limiting example of markerof resistance to at least one anti-viral useful with the viruses, can becited the mutations of the reverse transcriptase enzyme of the humanimmunodeficiency virus, conferring reduced sensitivity to the nucleosidereverse transcriptase inhibitors. Lastly, as a non-limiting example ofmarkers of resistance to at least one antifungal useful with the yeasts,can be cited the mutation of the enzyme 1-3-b-D-glucane synthase ofCandida albicans, conferring a reduced sensitivity to the echinocandins.As a further example, can be mentioned resistance to the azoleantifungals in Candida albicans, in particular resistance tofluconazole. The target of fluconazole is an enzyme, lanosteroldemethylase, involved in the synthesis of ergosterol, the mainconstituent of the fungal wall. Resistance to fluconazole can beassociated with the appearance of point mutations in the erg11 genecoding for lanosterol demethylase.

By determination of the virulence of a microorganism, is understood theevaluation of the pathogenic, harmful and virulent nature of themicroorganism. As non-limiting examples of virulence markers in thebacteria, can be cited PVL (Panton-Valentine Leukocidin), a cytolytictoxin with two synergistic components (Lukfet LukS), present inStaphylococcus aureus, which is one of the most virulent toxins causingskin involvements, extensive cellulitis, osteomyelitis and necrotisingpneumonias, and is involved in viral superinfections. Other examplescomprise Autolysin and Pneumolysin present in Streptococcus pneumoniae,a species responsible for infections of the respiratory tracts, formeningitis and for bacteremia, as well as the A and B toxins ofClostridium difficile, a commensal bacterium of the intestine, whicheither cause damage to the permeability of the intestinal epithelium (Atoxin) or directly attack the cells of the epithelium (B toxin), or intime reduce the intestinal transit and the intestinal absorption,causing diarrhoea (combined action of the A and B toxins). The Shigatoxins Stx1 and Stx2 present in Escherichia coli can also be cited as anexample. These two cytotoxins are considered as important virulencefactors of the enterohaemorrhagic Escherichia coli. They are responsiblefor complications such as haemorrhagic colitis or uremic haemolyticsyndrome. As a non-limiting virulence marker in the protozoans, can becited antioxidants (Fe-hydrogenase 2, peroxiredoxin, dismutasesuperoxide) present in Entamoeba histolytica, a species responsible fordysentery, hepatic abscess. As a non-limiting example of virulencemarkers in the viruses, can be cited the variant of the Nef protein inthe type-1 human immunodeficiency virus, the most pathogenic type in thehuman being. Lastly, as a non-limiting virulence marker in the yeasts,can be cited lipase 8 in Candida albicans, a species responsible forsuperficial candidiasis but also septicaemic and disseminatedcandidiasis. It should be noted that the specific virulence markers arealso usable as typing marker.

By way of example of bacteria being able to be characterised by ananalysis method of this type, can be cited:

-   -   Escherichia coli using TEM-2 as a resistance and typing marker,        as well as Shiga toxins, OmpA as a virulence and typing marker.    -   Enterococcus faecalis and faecium using VanA and VanB for        resistance and typing, as well as ESP (Enterococcal Surface        Protein) for virulence and typing, or    -   Staphylococcus aureus using the protein called immunoglobulin        G-binding protein A (also called protein A) for typing, the        protein PBP2a for resistance, or even typing, as well as the        protein PVL for virulence, or even also typing.

As other microorganisms able to be characterised by the above-mentionedanalysis method, can be cited:

-   -   Candida albicans using the enzyme 1-3-b-D-glucane synthase or        the enzyme lanosterol demethylase as a resistance and typing        marker, as well as lipase 8 as a virulence and typing marker.

The procaryotic and/or eucaryotic cells according to the presentinvention can be obtained from any sample able to contain a targetmicroorganism. The sample can be of biological origin, either animal,vegetable or human. It can then correspond to a biological fluid sample(whole blood, serum, plasma, urine, cerebrospinal fluid, organicsecretion, for example), a tissue sample or isolated cells. This samplecan be used as such to the extent that the characterisation markers ofthe microorganisms are available in the assayed sample, or it can besubjected, prior to analysis, to preparation of enrichment, extraction,concentration, purification, culture type, according to methods known tothe man skilled in the art.

The sample can be of industrial origin, or, according to anon-exhaustive list an air sample, water sample, sample taken from asurface, a piece or a manufactured product, a product of food origin. Ofthe samples of food origin, can be cited non-exhaustively a milk productsample (yoghurts, cheeses), meat, fish, egg, fruit, vegetable, water,beverage (milk, fruit juice, soda, etc.). These samples of food origincan also come from sauces or prepared dishes. A food sample can lastlycome from an animal feed, such as in particular animal meals.

The mass spectrometry to be employed in the method of analysis accordingto the invention is widely known to the man skilled in the art as apowerful tool for the analysis and the detection of different types ofmolecules. Generally, any type of molecule able to be ionised can bedetected as a function of its molecular mass by means of a massspectrometer. Depending on the nature of the molecule to be detected, ofprotein or metabolic origin, certain mass spectrometry technologies canbe more suited. Nevertheless, whatever the mass spectrometry method usedfor the detection, the latter comprises a step of ionisation of thetarget molecule into ions known as molecular, in the present case a stepof ionisation of the characterisation markers, and a step of separationof the molecular ions obtained as a function of their mass.

All mass spectrometers therefore include:

-   -   i) an ionisation source intended to ionise the markers present        in the sample to be analysed, i.e. to confer a positive or        negative charge to these markers;

ii) a mass analyser intended to separate the ionised markers, ormolecular ions 15, as a function of their mass over charge (m/z) ratio;

iii) a detector intended to measure the signal produced either directlyby the molecular ions, or by ions produced from the molecular ions, asdetailed below.

The ionisation step necessary for the use of a mass spectrometer can beperformed by any method known to the man skilled in the art. Theionisation source allows the molecules to be assayed to be brought intoa gaseous and ionised state. An ionisation source can be used either inpositive mode to study positive ions, or in negative mode to studynegative ions.

Several types of sources exist and will be used depending on therequired result and the molecules analysed. In particular can be cited:

-   -   electronic ionisation (EI), chemical ionisation (CI) and        desorption chemical ionisation (DCI)    -   bombardment by fast atoms (FAB), metastable atoms (MAB) or ions        (SIMS, LSIMS)    -   inductive plasma coupling (ICP)    -   chemical ionisation at atmospheric pressure (APCI) and        photoionisation at atmospheric pressure (APPI)    -   electronebulisation or electrospray (ESI)    -   laser-assisted desorption-ionisation by MALDI matrix,        surface-activated or on silicon    -   ionisation-desorption by interaction with metastable species        (DART).

In particular, the ionisation can be employed as follows: the samplecontaining the target molecules is introduced into an ionisation source,in which the molecules are ionised in the gaseous state and thustransformed into molecular ions which correspond to the initialmolecules. An ionisation source of electrospray type (ESI forElectroSpray Ionisation) permits ionisation of a molecule while causingit to pass from a liquid state to a gaseous state. The molecular ionsobtained then correspond to the molecules present in the liquid state,with in positive mode one, two, or even three additional protons ormore, and are therefore carriers of one, two, or even three charges ormore. For example when the target molecule is a protein, ionisation ofthe proteotypic peptides obtained after fractionation of the targetprotein, by means of a source of electrospray type operating in positivemode, leads to polypeptide ions in the gaseous state, with one, two, oreven three additional protons or more which are therefore carriers ofone, two, or even three charges or more, and permits passage from aliquid state to a gaseous state This type of source is particularly wellsuited, when the target molecules or proteotypic peptides obtained arepreviously separated by reverse-phase liquid chromatography.Nevertheless, the ionisation yield of the molecules present in thesample can vary depending on the concentration and the nature of thedifferent species present. This phenomenon results in a matrix effectwell known to the man skilled in the art.

A MALDI ionisation source will permit ionisation of the molecules, froma sample in the solid state.

The mass analyser in which the separation of the ionised markers as afunction of their mass/charge (m/z) ratio is performed is any massanalyser known to the man skilled in the art. Low resolution analysers,of quadripole or quadrupole (Q), 3D (IT) or linear (LIT) ion trap, alsocalled ionic trap, type and high resolution analysers, permittingmeasurement of the exact mass of the analytes and which use inparticular the magnetic sector coupled with an electric sector, the timeof flight (TOF) can be cited.

The separation of the molecular ions as a function of their m/z ratiocan be employed once (simple mass spectrometry or MS), or severalsuccessive MS separations can be performed. When two successive MSanalyses are performed, the analysis is called MS/MS or MS2. When threesuccessive MS separations are performed the analysis is called MS/MS/MSor MS3 and more generally, when n successive MS separations areperformed, the analysis is called MSn.

Of the techniques employing several successive separations, modes SRM(Selected Reaction Monitoring) in case of detection or assay of a singletarget molecule, or MRM (Multiple Reaction Monitoring) in case ofdetection or assay of several target molecules, are particular uses ofMS2 separation. Similarly MRM3 mode is a particular use of MS/MS/MSseparation. It is then called targeted mass spectrometry.

In the case of detection in simple MS mode, it is the mass/charge ratioof the molecular ions obtained which is correlated with the targetmolecule to be detected.

In the case of detection in MS/MS mode, essentially two steps are added,relative to an MS assay which are:

-   -   i) fragmentation of the molecular ions, then called precursor        ions, to give ions called 1st generation fragment ions, and    -   ii) separation of the ions called 1st generation fragment ions        as a function of their mass (m/z) 2, the ratio (m/z)1        corresponding to the (m/z) ratio of the precursor ions.

It is then the mass/charge ratio of the 1^(st) generation fragment ionsthus obtained which is correlated with the target molecule to bedetected. By first generation fragment ion, is understood an ionemanating from the precursor ion following a fragmentation step and themass over charge m/z ratio of which is different from the precursor ion.

The pairs (m/z)1 and (m/z)2 are termed transitions and arerepresentative of the characteristic ions to be detected.

The choice of the characteristic ions which are detected to becorrelated with the target molecule is made by the man skilled in theart according to standard methods. Their selection will advantageouslylead to the most sensitive, the most specific and the most robust assayspossible, in terms of reproducibility and reliability. In the methodsdeveloped for the selection of proteotypic (m/z)₁, and of firstgeneration fragment (m/z)₂ peptides, the choice is essentially based onthe intensity of the response. For more details, reference can be madeto V. Fusaro et al. Commercial software, such as the MIDAS and MRM Pilotsoftware by Applied Biosystems or MRMaid can be used by the man skilledin the art to allow him to predict all the possible transition pairs. Hecan also have recourse to a database called PeptideAtlas, constructed byF. Desiere et al. to compile all of the MRM transitions of peptidesdescribed by the scientific community. This PeptideAtlas base isavailable on a free access basis on the internet.

An alternative approach to select the proteotypic peptides (obtained forexample by tryptic digestion; cf. above) (m/z)₁ and (m/z)₂, consists inusing the MS/MS fragmentation spectra obtained during other works. Theseworks can be, for example, the phases of discovery and of identificationof the biomarkers by proteomic analysis. This approach has been proposedby Thermo Scientific at a users meeting It permits generation of a listof candidate transitions from peptides experimentally identified by theSIEVE software (Thermo Scientific). Certain criteria have been detailedby J. Mead et al. for the choice of (m/z)₁ and (m/z) 2 and are detailedbelow:

-   -   Peptides with internal cleavage sites, i.e. with internal Lysine        or Arginine, must be avoided, unless the Lysine or Arginine is        followed by Proline,    -   Peptides with Asparagine or Glutamine must be avoided as they        can deaminate,    -   Peptides with Glutamine or Glutamic Acid at the N-terminal must        be avoided as they can spontaneously form into a ring,    -   Peptides with Methionine must be avoided as they can be        oxidised,    -   Peptides with Cysteine must be avoided as they can be modified        in a non-reproducible manner at a possible step of denaturation,        reduction and locking of the thiol functions,    -   Peptides with Proline can be considered as favourable because        they generally produce intense fragments under MS/MS with a        single very major peak. However, a single very major fragment        does not permit validation of the identity of the transition in        a complex mixture. Indeed, only the simultaneous presence of        several characteristic fragments permits verification that the        precursor ion sought has indeed been detected,    -   Peptides having a Proline adjacent to the C-terminal (position        n−1) or in the second position relative to the C-terminal        (position n−2) are to be avoided as, in this case, the size of        the first generation peptide fragment is generally considered as        too small to be sufficiently specific,    -   The selection of fragments having a mass greater than the        precursor is to be preferred to promote specificity. For this        purpose, it is necessary to select a discharge precursor ion and        select the most intense first generation fragment ion having a        mass greater than the precursor, i.e. a first generation        fragment ion with a single charge.

The fragmentation of the selected precursor ions is performed in afragmentation cell such as models of triple quadripole type or of iontrap type or of time of flight (TOF) type which also permit theseparation of the ions. The fragmentation or fragmentations will beperformed conventionally by collision with an inert gas such as argon ornitrogen, in an electric field, by photo-excitation or photodissociationusing an intense light source, collision with electrons or radicalspecies, by application of a potential difference, for example in a timeof flight tube, or by any other method of activation. Thecharacteristics of the electric field determine the intensity and thenature of the fragmentation. Thus, the electric field applied in thepresence of an inert gas, for example in a quadripole, determines thecollision energy contributed to the ions. This collision energy will beoptimised, by the man skilled in the art, to increase the sensitivity ofthe transition to be assayed. As an example, it is possible to vary thecollision energy between 5 and 180 e⁻V under q2 in an AB SCIEX QTRAP®5500 mass spectrometer by the company Applied Biosystems (Foster City,United States of America). Similarly, the duration of the collision stepand the excitation energy in, for example, an ion trap will beoptimised, by the man skilled in the art to lead to the most sensitiveassay. As an example, it is possible to vary this duration, termedexcitation time, between 0.010 and 50 ms and the excitation energybetween 0 and 1 (arbitrary unit) under Q3 in an AB SCIEX QTRAP®5500 massspectrometer by the company Applied Biosystems.

Lastly, the detection of the selected characteristic ions is effected inconventional manner, in particular by means of a detector and of aprocessing system. The detector collects the ions and produces anelectrical signal the intensity of which depends on the quantity ofirons collected. The signal obtained is then amplified for it to be ableto be processed by computer technology. A computer technology unit forprocessing the data allows the data received by the detector to beconverted into a mass spectrum.

The principle of the SRM mode, or of the MRM mode, is to specificallyselect a precursor ion, to fragment it, and then to specifically selectone of its fragment ions. For such applications, devices of the triplequadripole or triple quadripole with ion trap hybrid type are generallyused.

In the case of a triple quadripole device (Q1q2Q3) used in MS² mode, forthe assay or the detection of a target protein, the first quadripole(Q1) permits filtration of the molecular ions, corresponding to theproteotypic peptides characteristic of the protein to be assayed andobtained at a prior digestion step, as a function of their mass overcharge (m/z) ratio. Only the peptides having the mass/charge ratio ofthe proteotypic peptide sought, ratio called (m/z)₁, are transmittedinto the second quadripole (q2) and act as precursor ions for thesubsequent fragmentation. The q2 analyser permits fragmentation of thepeptides of mass/charge ratio (m/z)₁ into first-generation fragmentions. The fragmentation is generally obtained by collision of theprecursor peptides with an inert gas, such as nitrogen or argon in q2.The first-generation fragment ions are transmitted into a thirdquadripole (Q3) which filters the first-generation fragment ions as afunction of a specific mass over charge ratio, which ratio is called(m/z)₂.

Only the first-generation fragment ions having the mass/charge ratio ofa fragment characteristic of the proteotypic peptide sought (m/z)₂ aretransmitted into the detector to be detected, or even quantified.

This mode of operation presents double selectivity, relative to theselection of the precursor ion on the one hand and selection of thefirst-generation fragment ion on the other. Mass spectrometry in SRM orMRM mode is therefore advantageous for quantification.

When the mass spectrometry employed in the method of the invention istandem mass spectrometry (MS2, MS3, MS4 or MS5), several mass analyserscan be linked together. For example, a first analyser separates theions, a collision cell allows fragmentation of the ions, and a secondanalyser separates the fragment ions. Certain analysers, such as iontraps or FT-ICR, constitute several analysers in one and permitfragmentation of the ions and analyse the fragments directly.

According to preferred embodiments of the invention, the method of theinvention comprises one or more of the following characteristics:

-   -   the mass spectrometry, employed for the properties of typing, of        potential resistance to at least one antimicrobial and virulence        factor, is spectrometry of MS/MS type, which has the advantage        of generating a specific fragment of the molecule to be detected        or to be quantified, and thus brings great specificity to the        assay method;    -   the MS/MS spectrometry is MRM, which has the advantage of using        an analysis cycle time in the mass spectrometer of some tens of        milliseconds, which permits detection or quantification with        high sensitivity, and in multiplexed manner, of a large number        of different molecules;    -   the determination of the properties of typing, resistance to an        antimicrobial and virulence factor is performed in the same mass        spectrometry apparatus, preferably simultaneously, which has the        advantage of reducing the analysis time and the cost of the        instrument, this also facilitates the processing and the        presentation of the results.

In addition to the determination of the properties of typing, resistanceto an antimicrobial and virulence factor, it is advisable to identifythe microorganism or microorganisms present in the sample to be assayed.

The methods of identification of microorganisms are widely known to theman skilled in the art, as described for example by Murray, P. R. et al.in Manuel of Clinical Microbiology, 2007, 9th edition, and in particularin Vol. I, Section III, chapters 15 and 16 for bacteria and yeasts, Vol.II, Section VI, chapter 82 for viruses, and Vol. II, Section X, chapter135 for protozoans. By way of example of conventional identificationmethods, the determination of the biological profile can be cited, usingfor example the Vitek 2 identification cards (bioMérieux), or usingtechniques of molecular biology with identification criteria based oninvestigating the presence of certain genes, and their sequence.

Identification can be performed directly from the sample in which theidentification is effected, or the microorganisms contained in thesample can be cultured by methods well-known to the man skilled in theart with optimal culture media and culture conditions suited to thespecies of microorganisms to be researched, as described by Murray P. R.et al. in Manuel of Clinical Microbiology, 2007, 9th edition, Vol. I,Section III, chapter 14, and in particular in Vol. I, Section IV,chapter 21 for bacteria, Vol. II, Section VI, chapter 81 for viruses,Vol. II, Section VIII, chapter 117 for yeasts, and Vol. II, Section X,chapter 134 for protozoans.

Thus, generally, in the case of identification by a biochemical methodof a bacterium in a sample, it is firstly necessary to obtain it in pureculture, for example after seeding on agar-agar. Molecular biology (PCR)can in certain cases be applied directly to the sample to be analysed.

Instead of culturing the microorganisms, the latter can be concentratedby capture directly from the sample by means of active surfaces. Such amethod has been described by W.-J.

Chen et al. who have captured different bacterial species by means ofmagnetic beads with a surface activated with Fe₃O₄/TiO₂. Capture byother means is also possible, such as capture by lectins or byantibodies or by Vancomycin Capture permits concentration of themicroorganisms and thus reduction or even elimination of the culturestep. This results in a considerable saving in time.

Identification can also be performed by mass spectrometry, according tothe techniques described above, preferably by MS, by MS/MS, or by MSfollowed by spectrometry of MS/MS type, which constitutes an embodimentof the invention. In this case also, the sample can be previouslysubjected to a culture step such as seeding on agar-agar.

The use of an identification method by MS is advantageous as it can beperformed in a few minutes and it requires a mass spectrometer with asingle analyser, i.e. a less complex instrument than a tandem massspectrometer used in MS/MS.

The use of an identification method by MS followed by spectrometry ofMS/MS type is also advantageous. It allows the identity of the ionsobserved under MS to be ascertained, which increases the specificity ofthe analysis.

The use of an identification method by MS/MS of MRM type has theadvantage of being more sensitive and more simple than the conventionalMS and then MS/MS approaches. This method requires neitherhigh-performance software to process the information between theacquisition of the MS spectrum and of the MS/MS spectrum, nor changingof the settings of the machine parameters to link the MS and then MS/MSspectra.

The identification method by MS can be performed with an electrospraysource on the raw sample, as described by S. Vaidyanathan et al. or byR. Everley et al. after chromatographic separation. Different m/z rangesthen permit identification of the organisms. S. Vaidyanathan et al. haveused a window between 200 and 2000 Th and R. Everley et al. a windowbetween 620 and 2450 Th. The mass spectra can also be deconvolved toaccess the mass of the proteins regardless of their state of charge. R.Everley et al. have thus exploited masses between approximately 5 000and 50 000 Da. Alternatively, the method of identification by MS canalso be performed by means of a MALDI-TOF, as described by Claydon et aland T. Krishnamurthy and P. Ross The analysis associates the acquisitionof a mass spectrum and the interpretation of expert software. It isextremely simple and can be performed in a few minutes. This method ofidentification is currently becoming widespread in medical analysislaboratories

The identification of bacteria by MS and then MS/MS via their proteinspresent in the sample has been widely applied by many teams. As anexample, it is possible to cite the recent works of Manes N. et al. whohave studied the peptidome of Salmonella enterica, or the works of R.Nandakumar et al. or of L. Hernychova et al. who have studied theproteome of bacteria after digestion of the proteins with trypsin. Theconventional approach consists in i) acquiring an MS spectrum, ii)successively selecting each precursor ion observed in the MS spectrumwith an intense signal, iii) successively fragmenting each precursor ionand acquiring its MS/MS spectrum, iv) interrogating protein databasessuch as SWISS-PROT or NCBI, through software such as Mascot (MatrixScience, London, United Kingdom) or SEQUEST (Thermo Scientific, Waltham,United States of America), to identify the peptide having a highprobability of corresponding to the MS/MS spectrum observed. This methodcan lead to the identification of a microorganism if a protein or apeptide characteristic of the species is identified.

According to yet another embodiment, the identification of said at leastone microorganism is performed by a conventional identification methodand the method of the invention comprises an additional step ofconfirmation of the identification of said at least one microorganism,which confirmation step is performed by mass spectrometry, according tothe techniques described above for the identification of microorganisms.

According to a particular embodiment, the mass spectrometry of theconfirmation step is mass spectrometry of MS/MS type, preferably MRM.

One of the advantages of the use of mass spectrometry resides in thefact that it is particularly useful for quantifying molecules, in thepresent case the markers of the properties of typing, resistance to atleast one antimicrobial. For this purpose, use is made of the detectedcurrent intensity, which is proportional to the quantity of the targetmolecule. The current intensity thus measured can serve for quantitativemeasurement permitting determination of the quantity of target moleculepresent, which is characterised by its expression in InternationalSystem of units (SI) of mol/m³ or kg/m³ type, or by multiples orsub-multiples of these units, or by the usual derivatives of the SIunits, including their multiples or sub-multiples. As a non-limitingexample, units such as ng/ml or fmol/l are units characterising aquantitative measurement.

Calibration is nevertheless necessary to be able to correlate themeasured area of the peak, corresponding to the current intensityinduced by the detected ions, with the quantity of target molecule to beassayed. For this purpose, the calibrations conventionally used in massspectrometry can be performed, within the framework of the invention.MRM assays are conventionally calibrated by means of external standardsor, preferably, by means of internal standards such as described by T.Fortin et al. In the case in which the target molecule is a proteotypicpeptide, permitting assay of a protein of interest, the correlationbetween the quantitative measurement and the quantity of targetproteotypic peptide, and therefore of protein of interest, is obtainedby calibrating the measured signal relative to a calibration signal forwhich the quantity to be assayed is known. The calibration can beperformed by means of a calibration curve, for example obtained bysuccessive injections of calibration proteotypic peptide at differentconcentrations (external calibration), or in preferred manner, byinternal calibration using a heavy peptide, as an internal standard, forexample according to the AQUA, QconCAT or PSAQ methods detailed below.By “heavy peptide” is understood a peptide corresponding to theproteotypic peptide, but in which one or more atoms of carbon 12 (¹²C)is (are) replaced by carbon 13 (¹³C), and/or one or more atoms ofnitrogen 14 (¹⁴N) is (are) replaced by nitrogen 15 (¹⁵N).

The use of heavy peptides, as internal standards (AQUA), has also beenproposed in patent application US 2004/0229283. The principle is toartificially synthesise proteotypic peptides with amino acids includingisotopes heavier than the usual natural isotopes. Such amino acids areobtained, for example, by replacing certain of the carbon 12 (¹²C) atomswith carbon 13 (¹³C), or by replacing certain of the nitrogen 14 (¹⁴N)atoms with nitrogen 15 (¹⁵N). The artificial (AQUA) thus synthesised hasexactly the same physicochemical properties as the natural peptide (withthe exception of a higher mass). It is generally added, at a givenconcentration, to the sample upstream of the assay by mass spectrometry,for example between the treatment causing cleavage of the proteins ofthe sample of interest and the fractionation of the peptides obtainedafter the treatment step. As a result, the AQUA peptide is co-purifiedwith the natural peptide to be assayed, on fractionation of thepeptides. The two peptides are therefore injected simultaneously intothe mass spectrometer, for assay. They are then subject to the sameionisation yields in the source. The comparison of the peak areas of thenatural peptides and AQUA, the concentration of which is known, permitscalculation of the concentration of the natural peptide therebydetermining the concentration of the protein to be assayed. Amodification of the AQUA technique has been proposed by J.-M. Pratt etal. under the name of QconCat. This modification is also described inpatent application WO 2006/128492. It consists in concatenatingdifferent AQUA peptides and producing the artificial polypeptide in theform of heavy recombinant protein. The recombinant protein issynthesised with amino acids including heavy isotopes. In this manner,it is possible to obtain a standard for calibrating the simultaneousassay of several proteins at lesser cost. The QconCAT standard is addedat the start, upstream of the treatment causing the cleaving of theproteins and before the steps of fractionation of the proteins, ofdenaturation, of reduction and then locking of the thiol functions ofthe proteins, if these are present. The QconCAT standard is thereforesubject to the same treatment cycle causing the cleaving of the proteinsas the natural protein, which permits taking into account the yield ofthe treatment step causing the cleaving of the proteins. Indeed, thetreatment, particularly by digestion, of the natural protein can beincomplete. In this case the use of an AQUA standard would lead to anunder-estimation of the quantity of natural protein. For an absoluteassay, it can therefore be important to take into account the yields ofthe treatment causing the cleaving of the proteins. However, V. Brun etal. have shown that, sometimes, the QconCAT standards did not exactlyreproduce the treatment yield, particularly by digestion of the naturalprotein, doubtless due to the fact of different three-dimensionalconformation of the QconCAT protein.

V. Brun et al. have then proposed the use of a method termed PSAQ anddescribed in patent application WO 2008/145763. In this case, theinternal standard is a recombinant protein, having the same sequence asthe natural protein but synthesised with heavy amino acids. Thesynthesis is performed ex-vivo with heavy amino acids. This standard hasstrictly the same physicochemical properties as the natural protein(with the exception of a higher mass). It is added at the start, beforethe step of fractionation of the proteins, when the latter is present.It is therefore co-purified with the native protein, at the step offractionation of the proteins. It has the same treatment yield,particularly by digestion, as the native protein. The heavy peptideobtained after cleavage is also co-purified with the natural peptide, ifa step of fractionation of the peptides is performed. The two peptidesare therefore injected simultaneously into the mass spectrometer, to bequantitatively assayed. They are subject to the same ionisation yieldsin the source. The comparison of peak areas of the natural peptides andof the reference peptides in the PSAQ method permits calculation of theconcentration of protein to be assayed taking into account all of thesteps of the assay method.

All of these techniques, i.e. AQUA, QconCAT or PSAQ or any othercalibration technique, used in assays by mass spectrometry and inparticular in MRM or MS assays, could be employed to perform thecalibration.

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The invention claimed is:
 1. Method of obtaining peptides fromprocaryotic and/or eucaryotic cells, said method comprising thefollowing steps: a) lysis of the procaryotic and/or eucaryotic cells ata low concentration of chaotropic agent(s) and recovery of the proteinsthus obtained, wherein the lysis at a low concentration of chaotropicagent(s) comprises lysis by sonication performed by means of anultrasound probe; b) denaturation of said proteins using at least onedenaturing agent, wherein the denaturing agent is selected from thegroup consisting of chaotropic agents, thiol reducing agents,detergents, and combinations thereof, and wherein the lysis anddenaturation steps are performed simultaneously; c) alkylation of thedenatured proteins using at least one alkylating agent; d) enzymaticproteolysis of the proteins obtained at the end of step c) using atleast one proteolytic enzyme; e) recovery of the peptides obtained atthe end of the enzymatic proteolysis step d); and wherein said method isperformed under a pressure below 100 bars.
 2. Method according to claim1, in which the lysis at a low concentration of chaotropic agent(s) isperformed at a concentration of chaotropic agent(s) less than or equalto 1M.
 3. Method according to claim 1, in which the lysis at a lowconcentration of chaotropic agent(s) is performed in the absence ofnon-saline chaotropic agent(s).
 4. Method according to claim 1, in whichsaid ultrasound probe is not subjected to a preheating step prior tolysis by sonication.
 5. Method according to claim 1, in which theproteolytic enzyme is a serine protease selected from the groupconsisting of trypsin, chymotrypsin and elastase.
 6. Method according toclaim 1, in which the denaturing agent is a thiol reducing agent. 7.Method according to claim 1, in which the alkylating agent is selectedfrom the group consisting of N-ethylmaleimide, iodoacetamide andM-biotin.
 8. Method according to claim 1, in which the minimum durationcorresponding to the step of enzymatic proteolysis in step d) isapproximately 15 minutes.
 9. Method according to claim 1, in which atleast steps a)-c) are performed simultaneously.
 10. Method according toclaim 1, in which steps a)-d) are performed simultaneously.
 11. Methodaccording to claim 9, in which: the denaturing agent is a thiol reducingagent selected from the group consisting of tris(2-carboxyethyl)phosphine and dithiothreitol, and the alkylating agent is iodoacetamide.12. Method according to claim 1, said method being performed underatmospheric pressure.
 13. Method of analysis of peptides of at least oneof procaryotic and eucaryotic cells comprising the following steps: i)Obtaining peptides from said procaryotic and/or eucaryotic cells usingthe method according to claim 1; and ii) Analysis of the peptides thusobtained, said analysis being performed using an analysis means of massspectrometry type.
 14. Method according to claim 1, said method beingperformed under a pressure of less than 50 bars.
 15. Method according toclaim 1, said method being performed under a pressure of less than 10bars.
 16. Method according to claim 1, in which the lysis at a lowconcentration of chaotropic agent(s) is performed at a concentration ofchaotropic agent(s) less than or equal to 100 mM.
 17. Method accordingto claim 1, in which the denaturing agent is a thiol reducing agentselected from the group consisting of 2-mercaptoethanol,tris(2-carboxyethyl)phosphine, dithioerythritol, tributylphosphine anddithiothreitol.